Ultrasensitive identification and precise quantitation of low frequency somatic m

描述(由申请人提供)：对癌症基因组中临床相关的体细胞变化的超灵敏检测在影响患者护理方面具有巨大的潜力，例如，在早期发现、确定诊断、改善预后、指导治疗和监测复发方面。然而，目前的技术不太适合对存在于极低频率的体细胞突变进行稳健的检测。大规模并行测序代表着一条前进的道路，但其检测非常罕见的事件的灵敏度从根本上受到测序错误率的限制。我们的目标是开发一种新的实验范式来克服这一限制。在我们的方法中，存在于样本中的靶序列的每个拷贝在多重捕获反应的第一个周期中用唯一的条形码序列被分子标记。扩增后，对靶扩增产物及其对应的条形码进行大规模并行测序。在分析过程中，条形码被用来关联共享相同起始点的序列读取。通过过度抽样，条形码相关的读数相互纠错，为每个前体分子产生一个独立的单倍体共识，即“分子计数”。此外，共同衍生的读数的折叠本质上纠正了扩增过程中任何等位基因特有的偏见，因此对突变等位基因频率的估计可以伴随着精确的置信限。在我们的第一个目标中，我们将开发实验方法和分析工具，通过分子计数对目标体细胞突变进行强有力的检测，在100,000个未突变拷贝的背景中，频率低至1个突变拷贝。在我们的第二个目标中，我们将开发三种超灵敏的多重分子计数分析，专门针对临床相关的癌症突变或基因小组，并严格评估它们的重复性。对于罕见的躯体事件的超灵敏、多路检测的强大、成本效益高、普遍适用的工具的可用性将是将癌症遗传学的发现转化为临床环境的变革性的一步。 与公共健康相关：随着我们进入“个性化医学”时代，DNA测序技术对公共健康将变得越来越重要，有助于解开人类疾病的遗传基础，也有助于临床诊断。这项提议旨在开发超灵敏的方法来检测肿瘤样本中与癌症相关的突变。这些技术有可能直接将癌症遗传学方面的发现转化为临床应用，如癌症的早期发现和对癌症复发的监测。

DESCRIPTION (provided by applicant): The ultrasensitive detection of clinically relevant somatic alterations in cancer genomes has great potential for impacting patient care, e.g. for early detection, establishing diagnoses, refining prognoses, guiding treatment, and monitoring recurrence. However, current technologies are poorly suited to the robust detection of somatic mutations present at very low frequencies. Massively parallel sequencing represents one path forward, but its sensitivity to detect very rare events is fundamentally constrained by the sequencing error rate. Our goal is to develop a new experimental paradigm that overcomes this limitation. In our approach, each copy of a target sequence that is present in a sample is molecularly tagged during the first cycle of a multiplex capture reaction with a unique barcode sequence. After amplification, target amplicons and their corresponding barcodes are subjected to massively parallel sequencing. During analysis, the barcodes are used to associate sequence reads sharing a common origin. Through oversampling, barcode-associated reads error-correct one another to yield an independent haploid consensus for each progenitor molecule, i.e. "molecular counting". Furthermore, the collapsing of commonly derived reads inherently corrects for any allele-specific bias during amplification, such that estimates of mutant allele frequency can be accompanied by precise confidence bounds. In our first aim, we will develop experimental methods and analytical tools that enable the robust detection of targeted somatic mutations via molecular counting to frequencies as low as 1 mutated copy in a background of 100,000 unmutated copies. In our second aim, we will develop three ultrasensitive, multiplex molecular counting assays that are specifically targeted at panels of clinically relevant cancer mutations or genes, and rigorously evaluate these for reproducibility. The availability of robust, cost-effective, generically applicable tools for the ultrasensitive, multiplex detection of rare somatic events will be a transformative step forward for the translation of discoveries in cancer genetics to a clinical setting. PUBLIC HEALTH RELEVANCE: As we enter an era of "personalized medicine", DNA sequencing technology will be increasingly important to public health, contributing towards the unraveling of the genetic basis of human disease, as well as for clinical diagnostics. This proposal aims to develop ultrasensitive methods for detecting cancer-relevant mutations in tumor samples. These technologies have the potential to directly enable the translation of discoveries made in cancer genetics to clinical applications such as the early detection of cancer and the monitoring of patients for cancer recurrence.